A success targeted nano delivery to lung cancer cells with multi-walled carbon nanotubes conjugated to bromocriptine

In this research, a new nano drug-based multi-walled carbon nanotubes (MWCNTs) was prepared and evaluated qualitatively. Bromocriptine (BRC) was conjugated to functionalized carbon nanotubes. Then, the CHNS, FT-IR, SEM, and RAMAN tests for characterization of the conjugated drug were done. The nanofluid-containing nano-drug was evaluated on lung cancer cells (A549 & QU-DB) and MRC5 by MTT and flow cytometry tests. Then, the gene expression studies of dopamine receptor genes were done before and after nano-drug treatment. After that, a western blotting test was carried out for further investigation of dopamine receptors protein production. Finally, Bax and Bcl-2 secretion were measured by the ELISA method in cells affected by MWCNTs-BRC Nf compared to untreated cells. The results showed that the nano-drug had a significant lethal effect on cancer cells, while it had no toxicity on MRC5. Also, the nano-drug could significantly induce apoptosis in lung cancer cells at a lower dose compared to the drug alone. In this study, a targeted nano-drug delivery system was designed, and its performance was evaluated based on neurotransmitter pathways, and the results showed that it may be useful in the treatment of lung cancer. However, additional studies on animal models are underway.

Drug conjugation, nano-drug characterization, and nanofluid preparation. In this step, 3 g of the drug and 1 g of chlorate MWCNT powder were added to 50 ml of dimethylformamide (DMF) solution. The solution was refluxed at 60 and 70 °C for 36 h and was extracted with tetrahydrofuran, methanol (96%), and ethanol, and then the precipitate was dried at room temperature. Elemental analysis of C-H-N-S, FT-IR, and RAMAN tests were conducted to ensure the binding of the drug and CNTs. Observation of the functionalized nanotubes with drug and nanotubes was done using a scanning electron microscope (SEM). For nanofluid preparation, 0.2 g of nano-drug powder, 6 ml of 96% ethanol, and 0.06 g of Arabic gum were added to 100 ml of deionized water. The mixture was stirred for 20 min and was placed in an ice container, and then, sonication was carried out for 20 min at 200 W 19 . Cell culture. Two human lung cancer cell lines QU-DB (human large cell carcinoma line), A549 (human adenocarcinoma lung cancer cell lines), and MRC5 (human non-malignant lung fibroblast) as a control normal cell line were applied from the National Cell Bank, Pasteur Institute, Iran. A549 and QU-DB cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) (Biosera, cat no: LMD1111, France), and MRC5 cell was cultured in RPMI1640 (Biosera, cat no: LMR1638, France) complemented with 10% Fetal Bovine serum, penicillin, and streptomycin antibiotics (Biosera, UK) and incubated at 37 °C with 5% CO2.
In vitro cytotoxicity and apoptosis assays. To investigate the effect of conjugated nano-drug formulation on enhancing the cytotoxicity outcomes, in vitro cytotoxicity profile of free BRC, functionalized MWCNTs Nf, and MWCNTs-BRC Nf using MTT assay in triplicate was conducted on A549, QU-DB, and MRC5 cell lines 20 . About 10 4 cells were seeded in each well of 96-well plates in DMEM containing 10% FBS, and the cell was allowed to adhere and achieve the suitable confluence overnight with 5% CO2 at 37 °C in a humid CO2 incubator. Next, the medium was removed, and cells were exposed to drugs for 24-48 h, regarding the culture condition with different equal doses for MWCNTs Nf, MWCNTs-BRC Nf (0, 20,40, 80, 100, 200, 400, 800, 1200, 1600, 2000 μg/ml), and free BRC (0, 18,36,72,144,288, μmol/l). Then, half-maximal inhibitory concentration (IC 50 ) values were estimated in different groups within 24 and 48 h with 95% confidence intervals (95% CI). To investigate the level of apoptosis and necrosis induced by active targeted MWCNTs-BRC Nf, according to IC 50 values in different groups of cell lines, treatments with equal appropriate concentration (50 μg/ml) was performed. In addition, regarding a previous study, treatments of BRC (72 μmol/l) and combination treatment of equivalent amounts of BRC (72 μmol/l) + F-MWCNTs Nf (120 μg/ml) in the fresh medium was performed. After 48 h, the cells were detached and washed with PBS, and then, suspended in 500 ml of 1X Binding buffer. Then, 10 μl of FITC-Annexin V was added to the cells, and subsequently, 10 μl propidium iodide (PI) solution was added. In genes before and after treatments, the total RNA was extracted from all treated cells (GeneAll ® Hybrid-RTM kit), and cDNA synthesis and real-time PCR analysis were performed for DRD1-DRD5 and β-Actin, as an internal control gene. Table 1 presents the used specific primers. According to IC 50 values with 95% CI in different groups, ELISA analysis was performed to analyze Bax and Bcl2 protein expression and the absorption of the standards and samples at a wavelength of 470 nm. Also, for each group, western blot analysis was performed for the evaluation of DRD1-DRD5 protein expression 21,22 . Transmission electron microscopy (TEM) study. After culturing A549 cancer cells and reaching the appropriate surface confluence, a cell culture flask was treated according to the IC 50 value of nano-drug (MWC-NTs-BRC Nf) for each ml of cell culture (50 μg/ml), and after 48 h, it was examined by TEM. Cell suspension of A549 cell line containing at least 2.5 × 10 6 cells was centrifuged for 15 min at 3000 rpm, and then, the primary fixation was performed, as previously described 23 . Statistical analysis. Statistical examinations were performed using Graph Pad Prism version 9.0.0. From triplicate conducted experiments, dose-response curves and IC 50 values with 95% confidence intervals (95% CI) in each group and cell line were estimated. One-way ANOVA and Tukey's post hoc test were used to compare the results of the groups. A P value less than 0.05 was considered significant. Furthermore, the rate of cell apoptosis and necrosis was analyzed by Flowojo software version 7.6. All results are expressed as mean ± standard deviation.

Results
Nano-drug characterization. Table 2 and Figs. 1, 2, 3, 4 show the results of C-H-N-S, FT-IR, and RAMAN tests, and also SEM observation related to the conjugation of the drug. The results of the elemental analysis confirmed amid binding of drugs to the CNTs because detectable amounts of nitrogen compared to carboxylated CNTs did not contain any nitrogen. Considering the mass balance between these N contents and conjugated CNT, the loading of 0.2 mmol BRC/g of MWCNTs-BRC was found.
The results of fourier-transform infrared spectroscopy (FTIR) spectrums of carboxylated and drug-functionalized nanotubes (Figs. 1, 2) indicated that the peak at 1659 cm −1 corresponded to an amide bond, which indicates the binding of the nano-tube to BRC.
The modifications of MWCNT samples were investigated by SEM. According to Fig. 3, (S1-S4) the drugfunctionalized nanotubes had a different morphology and appear rougher compared to the naked nanotubes, which confirms that MWCNTs were functionalized with drugs. According to samples, a significant change in diameter was observed. The average diameter of the functionalized MWCNTs (chlorated) was 26.3 nm, whereas it improved in MWCNTs-BRC Nf to about 32.6 nm.
Gene expression, western blot, and ELISA analysis. Western blot analysis was performed to in-vitro assess the relative protein levels of dopamine receptors (D1-D5) in normal and lung cancer cells (Fig. 10) (S5-S10). As shown in Figs. 11, 12, 13 and 14, DRD2 and DRD4 receptors were significantly higher than other DRDs in treated (free BRC and MWCNTs-BRC Nf) lung cancer cell lines compared to untreated groups. According to one-way ANOVA, there were no significant differences between the intensity of each relative protein level of DRD2 receptors in untreated lung cancer cells, as well as treated or untreated MRC5 normal cell line. The expression levels of DRD2 receptors were significantly higher in nano drug-treated lung cancer cell lines compared to treated or untreated MRC5 normal cell lines. Also, the expression levels of DRD2 in BRC-treated lung cancer cell lines were significantly higher compared to treated or untreated MRC5 normal cell lines. Importantly, the expression levels of the DRD2 nano drug-treated QU-DB lung cancer cell line were significantly higher compared to the A549 lung cancer cell line. The expression levels of DRD4 (control, free BRC, and MWCNTs-BRC Nf) in cancer cell lines were higher significantly compared to MRC5 groups, and the expression levels of DRD4 were significantly higher in QU-DB lung cancer cell lines compared to A549 cell lines. The expression levels of DRD3 in the control groups of all cancer and normal cell lines were not considerably different and were almost the same. The expression levels of DRD3 in QU-DB cells, which were treated with nano-drug, were significantly higher compared to nano drug-treated MRC5 cell line. ELISA results of Bax (Bcl-2 associated X protein) and Bcl2 (B-cell lymphoma 2) protein expression levels in A549, QU-DB, and MRC5 cell lines after 48 h showed that in BRC and conjugated nano-drug treatments, the levels of Bax and Bcl2 in the three groups of cell lines were different. Bax was positively correlated with the level of apoptosis, whereas Bcl-2 was negatively correlated with the level of apoptosis (Fig. 15).
The Bax expression levels were higher in BRC-and nano drug-treated A549 and QU-DB cells compared to control cells, but they were only significant in nano drug-treated cells. There were no differences in Bax and Bcl-2 expression levels in the MRC5-treated cells compared to the control groups, and they showed almost the same levels, and overall, Bax and Bcl-2 expression levels were markedly different compared to A549 and QU-DB cells 24 .
Transmission electron microscopy study. TEM results of MWCNTs-BRC Nf treatment are shown in Fig. 16 (S11, S12). The diameter of conjugated MWCNTs was about 25-40 nm. As sonication might cut nanotubes to shorter fragments, the average length of MWCNTs was about ∼ 500 nm. It has been reported that the uptake of carboxylated MWCNTs by HEK293, a non-phagocytic cell, occurs by two approaches, first direct penetration of single nanotubes via the plasma membrane, and second, through the uptake of MWCNTs cluster via endocytosis processes 25 . Due to their greater diameter, MWCNTs are capable of endosomal escape, piercing lysosomes, release into the cytoplasm, and interaction with the nucleus 26 . Short MWCNTs can penetrate the cell nucleus 25 .

Discussion
In this study, a specific agonist of DRDs, BRC was delivered to lung cancer cells with MWCNTs and showed a significant anti-proliferative effect on cancer cells and low toxicity on normal lung cells 6,12,27,28 . Previous studies support the concept that the D2R agonist due to the inhibition of cancer progression can be used as a therapeutic agent. Besides, the developing number of studies point to the interaction between dopamine and the immune system. DRDs can act as modulators in the activities of both cellular and humoral immune systems 29    The intensity of each target protein band was divided by the corresponding intensity of the β-actin band internal control and statistically normalized data were used to comparing between groups. All experiments were performed in triplicates, and the results have been presented as mean ± standard deviation.  34 . A decrease in the expression levels of DRD2 in NSCLC tissues in comparison to adjacent normal lung tissues was negatively associated with the tumor size and total survival. DRD2 inhibits NSCLC cell growth by blocking the NF-κB signaling pathway both in vitro and in vivo 35 . Previous studies have shown that BRC as a specific agonist of DRDs exerted anticancer effects 6,12,13,[36][37][38] . In this research, after treatment of lung cancer cells with BRC, high expression levels of DRD2 were found and at a low dosage of the drug in conjugated form, this effect was found to be stronger while having more potent anti-cancer effects and less toxicity.
The MTT results after 24 h of treatment with free BRC, MWCNT NFs, and targeted MWCNTs-BRC Nf showed more sensitivity to apoptosis in A549 and QU-DB cells compared to normal MRC5 cell line. Furthermore, the targeted NPs significantly induce more apoptosis and less viability compared to free BRC and MWC-NTs. The results of the MTT assay after 48 h showed a more potent cytotoxicity effect of conjugated NPs than 24 h in both cancer cell lines compared to free BRC and F-MWCNTs Nf. A decrease in the IC 50 value of targeted MWCNTs-BRC Nf formulation was estimated in lung cancer cells compared to the MRC5 cell line after 24 and 48 h. According to the results, MWCNTs-BRC Nf had a significant effect on decreasing the viability rate and IC 50 values of cancer cell lines compared to the normal MRC5 cell line. This is due to the notable consequence of the DRD2s targeting in internalization of BRC into the cancer cell lines in addition to several parameters, such as high vehicle capacity, increase in the solubility and stability of functionalized MWCNTs, and needle-like structure of CNTs to cross the cell membrane 39,40 .
Accordingly, it can be concluded that the normal MRC5 cell line was significantly more resistant to apoptosis than A549 and QU-DB lung cancer cell lines and showed a higher viability rate against exposure to three different drugs. ELISA results of the relative expression levels of Bax and Bcl2 proteins confirmed our observations. Data analysis demonstrated increased Bax protein levels in lung cancer cell lines compared to MRC5, whereas decreased Bcl2 protein levels in lung cancer cell lines compared to MRC5 cells were observed. This could be due to the low expression of DRD2s on the MRC5 cell surface, which causes less interaction and drug uptake into the cell. Therefore, BRC as an agonist of DRDs conjugated by MWCNT NPs could have a synergic effect in lung cancer cells, making DRDs sensitive to programmed death, preventing their entry into the cell cycle, finally improving the cytotoxic effects.
Furthermore, it has been reported that cancer cells cause greater internalization of nanoparticles compared to normal cells because of the enhanced permeation and retention (EPR) effect 41 ; therefore, because of the vascular permeability of tumors, nanoparticles are accumulated in the tumor cells compared to normal cells 42,43 . Several    44,45 . DRD1-D5 mRNA and protein levels were detected by qRT-PCR and western blot analysis, respectively in all three cell lines. We found that DRD2 expression levels in lung cancer cell lines and normal MRC5 were nearly the same before treatment; nevertheless, after cell treatment with drugs, the expression levels of this receptor altered and when it was exposed to BRC, the expression levels of this receptor in cancer cells compared to normal MRC5 increased significantly and after treatment with targeted nano drag, particularly in QU-DB cells, a further increase was observed. Regarding DRD4, the expression levels were significantly higher in cancer cells than in normal cell  www.nature.com/scientificreports/ lines. After treatment with BRC and targeted nano-drug, increased expression levels were observed in both cancer cell lines; however, it was significant in QU-DB cells. Concerning the expression levels of receptor 3 before and after treatment, no significant difference was observed in cancer cells compared to normal cells, except in QU-DB cells that were exposed to targeted nano drag. Also, the expression levels of DRD1 and DRD5 genes did not show any significant changes. Our observations are in agreement with previous studies about the expression levels of DRDs and the role of their agonist in lung cancer 27,35,46 . Accordingly, it is critical to identify the exact pharmacological and toxicological properties of CNTs before utilizing them in medical settings. Surface functionalization and modification of CNTs are appropriate methods to reduce their cytotoxicity, overcome limitations, such as distribution, accumulation rate, and clear pathways for CNT-based drug delivery systems. Hence, approaches for surface functionalization comprising covalent and noncovalent functionalization are employed to synthesize CNTs 47 . Functionalization is also helpful in conjugating the therapeutic molecule or the ligands on the surface or ends of CNTs to make them active against cancer cells 40 . In this study, we demonstrated that CNT-based drug delivery possesses a magnificent potential for cancer therapy. Two basic properties have made carbon nanotubes suitable agents, especially for use and clinical purposes in drug delivery. One of these features is the ability to penetrate cell walls and increase drug load without causing cell death. Another feature is their biocompatibility so that after drug delivery, nanotubes can be eliminated from the systemic circulation through the renal excretory pathway without side effects [48][49][50] .
Herein, we reported the first active targeted, ligand-receptor BRC-conjugated MWCNTs and demonstrated in vitro killing of cancer cells, enhanced anticancer effects, and reduced cytotoxicity of the drug at a high dose in the normal cell line. Targeted drug delivery systems can improve the protection of healthy cells from the cytotoxic compounds, minimize drug threshold dose and inhibit adverse effects, and reduce the drug-resistant cancerous cells 51 .
In conclusion, in our previous study, it was found that the combination treatment with BRC and MWCNTs nano-fluid (MWCNTs Nf) could inhibit the proliferation of A549 and QU-DB cells through the induction of apoptosis in addition to necrotic effects. The present study was designed to further investigate the mechanism of suppression of lung cancer cell growth, as well as eliminate the necrotic effects, which seemed to be due to the synergistic and excessive effects of these compounds. To our knowledge, for the first time, we designed an active targeted BRC-conjugated functionalized MWCNTs nanofluid drug delivery system to investigate its efficiency on lung cancer cell lines. In this article, the functionalized MWCNTs Nf as a drug delivery system could improve the efficiency of the anti-tumor activity as well as reduce drug dosage and eventually decrease the side effects of BRC. www.nature.com/scientificreports/ Reprints and permissions information is available at www.nature.com/reprints.
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